Levels of histone modification in promoter of NOD-like receptor family pyrin domain-containing protein 3 in synovial fibroblasts of osteoarthritis

doi:10.12307/2022.596

Abstract

Abstract: BACKGROUND: 1,25(OH)2D3 deficiency can exacerbate osteoarthritis. Previous studies have shown that 1,25(OH)2D3 regulates the proliferation and apoptosis of synovial fibroblasts in arthritis, but has unclear effect on the regulation of inflammasome pathway in synovial fibroblasts.
OBJECTIVE: To determine the expression level of NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome pathway in osteoarthritic synovial fibroblasts and to explore the effect of 1,25(OH)2D3 on regulating NLRP3 inflammasome pathway and its specific mechanism.
METHODS: Synovial tissue samples from patients with or without osteoarthritis were clinical collected for primary cell culture. Protein and mRNA expression levels of NLRP3, caspase recruitment domain and Caspase-1 in primary synovial fibroblasts were detected. Additionally, primary synovial fibroblasts were stimulated by 1,25(OH)2D3 in vitro for 24 hours. The protein expression of NLRP3, caspase recruitment domain and Caspase-1 was then detected. The levels of H3K4me3, H2AK119Ub, and H3K27me3 in the promoter region of NLRP3 were detected by chromatin immunoprecipitation technique. Small interfering RNA technique was applied to knock down the expression level of vitamin D receptor in synovial fibroblasts, and the primary cells were stimulated by 1,25(OH)2D3 in vitro for 24 hours. Subsequently, the protein expression of NLRP3, caspase recruitment domain, and Caspase-1 was detected.
RESULTS AND CONCLUSION: Compared with non-osteoarthritis patients, NLRP3 inflammasome pathway associated proteins (NLRP3, caspase recruitment domain, and Caspase-1) were overexpressed in synovial fibroblasts of osteoarthritis patients, and the transcription level of NLRP3 in osteoarthritis patients was significantly higher than that in non-osteoarthritis patients (P < 0.01). Compared with non-osteoarthritis patients, 1,25(OH)2D3 significantly inhibited the transcription level of NLRP3 and activation of NLRP3 inflammasome pathway in synovial fibroblasts of osteoarthritis patients in vitro (P < 0.05). Knockdown of vitamin D receptor could attenuate the inhibitory effect of 1,25(OH)2D3 on NLRP3 transcription and abate its inhibitory effect on the activation of NLRP3 inflammasome pathway. These findings suggest that 1,25(OH)2D3 exerts an inhibitory effect on NLRP3 transcriptional expression mainly by upregulating the levels of H3K27me3 and H2AK119Ub and inhibiting the level of H3K4me3 in NLRP3 promoter region.

Key words: osteoarthritis, synovial fibroblast, inflammation, 1,25(OH)2D3, NLRP3, vitamin D receptor, histone modification

CLC Number:

Wang Jian, Gu Sanjun, Liu Yu, Zhao Kai, Li Haifeng. Levels of histone modification in promoter of NOD-like receptor family pyrin domain-containing protein 3 in synovial fibroblasts of osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(15): 2387-2393.

Figures/Tables 4

2.3 1，25(OH)2D3抑制滑膜成纤维细胞样细胞中NLRP3信号通路依赖于维生素D受体分子 1，25(OH)2D3调控下游靶点主要是通过与维生素D受体结合后发挥转录调控作用[24]。为了进一步探索1，25(OH)2D3调控NLRP3通路是否依赖于维生素D受体，通过使用小干扰RNA敲低滑膜成纤维细胞样细胞中维生素D受体的水平。通过免疫印迹检测NLRP3、Caspase招募域及Caspase-1的表达水平，结果发现，敲低滑膜成纤维细胞样细胞内维生素D受体的表达后，1，25(OH)2D3处理无法抑制滑膜成纤维细胞样细胞内NLRP3、Caspase招募域及Caspase-1分子的蛋白表达水平，这提示1，25(OH)2D3抑制滑膜成纤维细胞样细胞中NLRP3信号通路依赖于维生素D受体分子。为了进一步明确1，25(OH)2D3抑制NLRP3转录表达是否依赖于维生素D受体，通过实时荧光定量PCR实验发现，转染敲低维生素D受体之后，可以减少滑膜成纤维细胞样细胞内维生素D受体的表达水平，但是1，25(OH)2D3处理对滑膜成纤维细胞样细胞的维生素D受体表达似乎没有明显影响。与1，25(OH)2D3组相比，维生素D受体敲低后1，25(OH)2D3抑制骨关节炎患者滑膜成纤维细胞样细胞中NLRP3表达作用明显减弱(n=3，P < 0.001)；与对照组相比，维生素D受体敲低后，1，25(OH)2D3的抑制作用两者之间差异无显著性意义(n=3，P=0.1478)，提示1，25(OH)2D3的抑制作用可能主要依赖于维生素D受体。同时，检测Caspase招募域分子，发现维生素D受体敲除也可以减弱1，25(OH)2D3的抑制作用(n=3，P < 0.001)；检测Caspase-1分子也获得了同样的结论(n=3，P < 0.01)；与对照组相比，1，25(OH)2D3抑制Caspase-1的作用，两者之间差异无显著性意义(n=3，P=0.0887)。见图3A-E。通过实时荧光定量PCR实验结果发现，与si-NC组相比，敲低维生素D受体表达后1，25(OH)2D3抑制滑膜成纤维细胞样细胞NLRP3转录表达的作用减弱(n=3，P < 0.001)，且与对照组之间无明显差异(n=3，P=0.2587)，见图3F。综合上述结果，可以初步明确1，25(OH)2D3主要通过与维生素D受体结合后抑制NLRP3转录表达及下游信号通路的活化。"

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